Biotechnology centers on cloning and subcloning of genes, both of which are now routine procedures. The cloning step involves the creation of a gene library by fragmenting or restricting, using suitable restriction enzymes, the genome of an organism which comprises the starting material for the sought-after gene. These DNA fragments are then inserted into a suitable cloning vector, and the vector introduced into a host cell such as bacteria, yeast or like organism. Using appropriate detection and isolation techniques, host cells harboring vectors with a gene of interest can be identified, separated, and grown up in large amounts.
Regardless of the type of cloning vector employed, they are designed to facilitate creation of gene libraries by receiving large DNA fragments, and to be compatible with suitable host cells, as well as the materials and methods for growing up, isolating and detecting the vector in the host cells. Thus, particular cloning vectors generally have associated with them ancilliary biochemical materials and methods that are suited to the cloning process. A good example of this type of vector is bacteriophage lambda. Over the years its molecular biology has been elucidated, and consequently a number of vectors based on lambda, as well as support systems for isolating and propagating these vectors have been established.
Cloning a particular gene is generally just the initial step in isolating the gene in order to carry out specific biotechnical procedures utilizing the gene. Hence, it is often necessary to "subclone" the gene into a more functionally specific second vector. The latter are generally plasmids or derivatives or hybrids thereof. The process of subcloning consists of removing the DNA sequence from the initial cloning vector (i.e. lambda) with suitable restriction enzymes, and then inserting the DNA into a plasmid. Since the process of subcloning is time consuming, and technically arduous, the construction of high efficiency cloning vectors that would eliminate the subcloning step is a much sought after goal in biotechnology.
The ideal cloning vector would be transferable into bacteria and higher cells, and have the properties of both cloning and subcloning vectors. It can be imagined that this type of vector might consist of a subcloning vector integrated into the cloning vector, and that could be exised from the cloning vector to yield a plasmid. DNA could then be cloned directly into the subcloning vector, and the latter propagated and selected along with the cloning vector. Assuming that a method for excising and circularizing the subcloning vector from the cloning vector could be developed, the end result would be a vector that could be propagated, detected, and isolated using standard cloning vector techniques, yet bypass having to transfer DNA in a separate step into a subcloning vector, by excising and circularizing the subcloning vector.
It may be possible to generate the ideal cloning vector by manipulating particular DNA origins of replication. A common feature generally shared by cloning vectors is the presence of one or more DNA origins of replication. The latter allows for replication of the vector either autonomously, or concomitantly with host cell chromosomal DNA. Research efforts over the past several years have yielded information concerning nucleotide sequences that comprise origins of replication of different organisms. Further, work aimed at establishing which nucleotides in the origin are necessary for DNA synthesis has led to the realization that origins of replication, in at least some organisms, have distinct functional nucleotide domains. For instance, Dotto, et al in Journal of Molecular Biology (1984) Vol. 172, p. 507-521 have shown that the DNA origin of replication in filamentous single-stranded DNA bacteriophages comprises nucleotide sequences responsible for both initiating and terminating DNA synthesis. Thus, it may be possible to create the ideal vector consisting of a cloning vector, such as lambda, containing the initiator and terminator regions separated by intervening DNA. The intervening DNA could have numerous functions, including acting as a region into which to clone DNA. If conditions could be established whereby the initiator-terminator regions with accompaning intervening DNA could be excised from lambda with rejoining of the initiator-terminator regions, the end result would be a vector having the properties of a cloning vector (i.e. lambda), and an excisable plasmid (initiator-terminator regions and intervening DNA).
Although there exists a variety of DNA cloning techniques and vectors, there is a demand for cloning vehicles that facilitate a determination of the relative positions of DNA sequences along large stretches of DNA. For instance, it is generally difficult, or impossible to incorporate more than 50 kilobases into the bacteriophage vector, lambda. The latter is perhaps the most widely used and convenient vector for cloning large DNA fragments, because of the high efficiency with which lambda can be made to introduce genes into bacterial cells. Consequently this has led to the development of what is known as "chromosome walking" which involves isolating one recombinant phage or other cloning vehicle, particularly cosmids, and using it to isolate other recombinant vectors that contain overlapping DNA sequences. The technique relies on isolating a probe that can be used to identify a segment of DNA that is common to both a first and a second recombinant vector wherein the second recombinant contains additional and overlapping genetic information. The second recombinant can then be used to screen for a third recombinant containing information common to the second and third recombinants, and this procedure repeated to yield overlapping cloned gene segments. Unfortunately, however, "chromosome walking" is extremely time consuming and technically very demanding. In part, this is because present cloning systems do not facilitate cloning large segments of DNA. Thus a cloning vehicle that permits a determination of the relative positions of DNA sequences along large stretches of DNA is sorely needed.